Assembly of a Micro-Hotspot of Caenogastropod Endemism in the Southern Nevada Desert, with a Description of a New Species of Tryonia (Truncatelloidea, Cochliopidae)

A peer-reviewed open-access journal ZooKeys Assembly492: 107–122 of (2015)a micro-hotspot of caenogastropod endemism in the southern Nevada desert... 107 doi: 10.3897/zookeys.492.9246 RESEARCH ARTICLE http://zookeys.pensoft.net Launched to accelerate biodiversity research

Assembly of a micro-hotspot of caenogastropod endemism in the southern Nevada desert, with a description of a new species of Tryonia (Truncatelloidea, Cochliopidae)

Robert Hershler1, Hsiu-Ping Liu2, Jeffrey S. Simpson2

1 Department of Invertebrate Zoology, Smithsonian Institution, P.O. Box 37012, Washington, DC 20013- 7012, USA 2 Department of Biology, Metropolitan State University of Denver, Denver, CO 80217, USA

Corresponding author: Robert Hershler ([email protected])

Academic editor: T. Backeljau | Received 13 January 2015 | Accepted 16 March 2015 | Published 30 March 2015

http://zoobank.org/8C3C5DD3-7424-49EC-9444-E07917D82DBE

Citation: Hershler R, Liu H-P, Simpson JS (2015) Assembly of a micro-hotspot of caenogastropod endemism in the southern Nevada desert, with a description of a new species of Tryonia (Truncatelloidea, Cochliopidae). ZooKeys 492: 107–122. doi: 10.3897/zookeys.492.9246

Abstract Newly obtained and previously published sequences of the cytochrome c oxidase subunit I (COI) gene were analyzed to examine the biogeographic assembly of the caenogastropod fauna (belonging to the families Assimineidae, Cochliopidae, and Hydrobiidae) of an isolated spring along the lower Colorado River in southern Nevada (Blue Point Spring). Based on available COI clock calibrations, the three lineages that comprise this fauna are 2.78–1.42 million years old, which is roughly coeval or slightly younger than the age of Blue Point Spring (inferred from local fossil spring deposits). Two of the lineages—endemic Pyrgu- lopsis coloradensis and Assiminea aff.infima —are most closely related to snails in the Death Valley area (well to the west) and likely colonized Blue Point Spring by transport on birds. A single haplotype was detected in both of these snails, suggesting that they may have only recently colonized Blue Point Spring. The third lineage—endemic Tryonia infernalis, newly described herein based on morphological and molecular evidence—is most closely related to a geographically proximal species in a lower Colorado River tributary (T. clathrata); the split between these taxa may be the product of vicariance (severance of a prior drainage connection) or a separate jump dispersal event. The considerable genetic diversity inT. infernalis (three haplotypes differing by 0.6% mean sequence divergence) suggests a possibly lengthy history of local differentiation. Our findings also identify Blue Point Spring as a new micro-hotspot of groundwater-dependent biodiversity in Nevada and will assist ongoing efforts to protect and conserve these imperiled ecosystems.

Copyright Robert Hershler et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 108 Robert Hershler et al. / ZooKeys 492: 107–122 (2015)

Keywords Gastropoda, Assimineidae, Hydrobiidae, western United States, aquatic snails, biogeography, taxonomy, conservation

Introduction

The desert region of southeastern California and southwestern Nevada, encompassing portions of the Great Basin and lower Colorado River watershed, contains distinctive assemblages of tiny caenogastropods—belonging to the families Assimineidae (genus Assiminea), Cochliopidae (Tryonia) and Hydrobiidae (Pyrgulopsis)—that have been a recent focus of biogeographic study using mtDNA sequence data (e.g., Hershler et al. 1999a, b, Hershler and Liu 2008a, b). These assemblages broadly overlap geographically and are tightly linked with spring habitats; their biogeographic histories do not well correlate with surface drainage and have likely been shaped, at least in part, by overland dispersal on waterfowl (e.g., Liu et al. 2003, Hershler et al. 2005, Liu and Hershler 2007, Hershler and Liu 2008a). The biogeographic patterns of these assemblages also differ in important respects. The regional assimineids (referred to herein as the Assiminea infima complex) are amphibious animals that typically live on riparian vegetation along the margins of springs and spring runs. This assemblage belongs to a single lineage that diverged from marine (Pacific) coastal progenitors during the late Pliocene (Hershler and Liu 2008a). The other two assemblages are entirely aquatic: Tryonia is restricted to thermal waters while Pyrgulopsis lives in ambient temperature and thermal habitats. Both of these assemblages are composed of multiple lineages, some having long histories of diversification within the region (Hershler et al. 1999a, Hershler et al. 2011). The Tryonia assemblage is composed of a few subgroups that have close relationships with congeners from the lower Colorado River basin (T. angulata Hershler), northern Great Basin and western California (T. margae Hershler, T. salina Hershler), and northeastern Mexico (T. porrecta [Mighels, 1845]; clade composed of T. elata Hershler, T. ericae Hershler, T. variegata Hershler) (Hershler et al. 1999a, Hershler et al. 2011). ThePyrgulopsis assemblage contains a much larger number of lineages which have close relationships to taxa from western California, the lower Colorado River basin, and other portions of western North America (Hershler and Liu 2008b, Hershler et al. 2013). The A. infima complex is subdivided into a clade that is distributed in the Death Valley region (this lineage also contains a population from the head of the Gulf of California) and a genetically divergent population (A. aff.infima Berry) in Blue Point Spring (Hershler and Liu 2008a), which is located along Lake Mead ca. 150 km to the east-southeast (Fig. 1). Blue Point Spring also contains an endemic species of Pyrgulop- sis (P. coloradensis Hershler) and a population of Tryonia that was previously assigned to widely ranging T. porrecta (Hershler 2001), neither of which have been previously studied genetically. Here we analyze newly obtained and previously published DNA sequences to examine the intersection of the biogeographic histories of the three con- Assembly of a micro-hotspot of caenogastropod endemism in the southern Nevada desert... 109

Figure 1. Map showing the location of Blue Point Spring relative to other geographic areas discussed in the text. The collecting localities for specimens ofPyrgulopsis sanchezi and Tryonia clathrata (sister taxa of Blue Point Spring endemics) used in the molecular phylogenetic analyses are also shown. trasting groups of snails at this isolated spring. We also describe the Blue Point Tryonia as a new, endemic species based on molecular and morphologic evidence. Our results reveal a complex historic assembly of the Blue Point Spring snail fauna; and delineate this water body as another micro-hotspot of groundwater-dependent biodiversity in the region, which will assist ongoing efforts to protect and conserve these imperiled ecosystems (Greenwald and Bradley 2008, Abele 2011).

Methods

Fresh material was collected from Blue Point Spring by RH in May, 2014, and preserved in 90% ethanol for genetic analysis; a portion of the T. porrecta sample was relaxed with menthol crystals, fixed in dilute (4%) formalin, and preserved in 70% ethanol for anatomical study. Genomic DNA was extracted from entire snails (A. aff. infima, six 110 Robert Hershler et al. / ZooKeys 492: 107–122 (2015) specimens; P. coloradensis, four specimens; T. porrecta, six specimens) using a CTAB pro- tocol (Bucklin 1992); each specimen was analyzed for mtDNA separately. LCO1490 and HCO2198 (Folmer et al. 1994) were used to amplify a 710 base pair (bp) frag- ment of the cytochrome c oxidase subunit I gene (COI). Amplification conditions and sequencing of amplified polymerase chain reaction product were those of Liu et al. (2003). Sequences were determined for both strands and then edited and aligned using SEQUENCHERTM version 5.0.1. Novel haplotypes were not detected in the newly sequenced specimens of A. aff. infima and thus we did not update our previously published phylogenetic analysis of the A. infima complex (Hershler and Liu 2008a). The newly sequenced specimens of Blue Point Spring Tryonia were analyzed together with previously published sequences from 30 congeners and closely related Minckleyella balnearis Hershler, Liu & Landye (a monotypic genus from northern Mexico), with Mex- ipyrgus carranzae Taylor used to root the phylogenetic tree (per Liu et al. 2001). Given that Pyrgulopsis is a large genus containing 139 species (Hershler et al. 2014), most of which have been previously sequenced, we restricted our analysis of the relationships of P. coloradensis to the newly sequenced specimens from Blue Point Spring, and sequences of 18 congeners from adjacent areas (including those that were found to be most similar to the newly obtained haplotypes using a BLAST search) to obtain a readable tree. The phylogenetic tree for this dataset was rooted with Floridobia winkleyi (Pilsbry) (per Her- shler et al. 2003). One example of each haplotype detected in a given sample was used in the phylogenetic analyses. The new sequences from Blue Point Spring populations were deposited in GenBank (accession numbers KP899916–KP899919). MRMODELTEST 2.3 (Nylander 2004) was used to obtain an appropriate sub- stitution model (using the Akaike Information Criterion) and parameter values for the molecular phylogenetic analyses. MRMODELTEST selected GTR + I + G model parameters as the best fit model for both the Tryonia and Pyrgulopsis datasets. Phylogenetic analyses were performed using four different methodologies—distance, maximum parsimony (MP), maximum likelihood (ML) and Bayesian inference. The distance, MP, and ML analyses were performed using PAUP*4.ob10 (Swofford 2002), and the Bayesian analyses were conducted using MRBAYES 3.2.3 (Ronquist and Huelsenbeck 2003). For the distance analyses, GTR distance was used to generate a neighbor-joining (NJ) tree (Saitou and Nei 1987). The MP analyses were conducted with equal weighting, using the heuristic search option with tree bisection reconnection branch-swapping and 100 random additions. The ML analyses were performed using GTR + I + G model. A GTR distance based NJ tree was used as the initial topology for branch-swapping. Node support was evaluated by 10,000 bootstrap pseudo-replicates except for the ML analysis, in which support values were based on 100 replications. For the Bayesian analyses Metropolis-coupled Markov chain Monte Carlo simulations were run with four chains (using the model selected through MRMODELTEST) for 3,000,000 generations for Tryonia, and 2,000,000 generations for Pyrgulopsis. Markov chains were sampled at intervals of 10 generations to obtain 300,000 and 200,000 sample points, respectively. We used the default settings for the priors on topologies and the GTR + I + G model parameters selected by MRMODELTEST as the best fit model for both analyses. At the Assembly of a micro-hotspot of caenogastropod endemism in the southern Nevada desert... 111 end of the analyses, the average standard deviation of split frequencies was less than 0.01 (0.0036 and 0.0033, respectively) and the Potential Scale Reduction Factor (PSRF) was 1, indicating that the runs had reached convergence. The sampled trees with branch lengths were used to generate a 50% majority rule consensus tree, with the first 25% of the samples removed to ensure that the chain sampled a stationary portion. Genetic distances within and between samples were calculated using MEGA6 (Ta- mura et al. 2013), with standard errors estimated by 1,000 bootstrap replications with pairwise deletion of missing data. Since MEGA does not contain the GTR model that was selected by MRMODELTEST, we used the maximum composite likelihood distance, which is the nearest model. Tajima relative rate tests of local clock-like behavior (Tajima 1993) were performed using MEGA6. The posterior Bayes factor was used to test a global clock assumption (MRBAYES 3.2.3). Large, adult females were used for shell measurements. The total number of shell whorls (WH) was counted for each specimen; and the height and width of the entire shell (SH, SW), body whorl (HBW, WBW), and aperture (AH, AW) were measured from camera lucida outline drawings using a digitizing pad (see Hershler 1989). In addition, three ratios were generated from the raw data (SW/SH, HBW/SH, AH/SH). Descriptive statistics were generated using Systat for Windows 11.00.01 (SSI 2004). Sexual dimorphism in shells, which is commonly observed in Tryonia species (Taylor 1987), could not be quantified owing to the small sample size. Variation in the number of cusps on the radular teeth (n = 5) was assessed using the method of Hershler et al. (2007a). Descriptive terminology follows that of Taylor (1987) and Hershler (2001). The brief taxonomic description of the new species focuses on diagnostic fea- tures of external morphology. Types and other voucher material were deposited in the Smithsonian Institution’s National Museum of Natural History (USNM) collection.

Results

The phylogenetic analyses congruently depicted a sister relationship between P. coloradensis and P. sanchezi Hershler, Liu & Bradford, which is distributed in the Death Valley area (Fig. 1). This relationship was strongly supported (1.0 posterior probabil- ity) only in the Bayesian analysis (the Bayesian tree is shown in Fig. 2). This clade in turn was depicted as sister to P. deserta (Pilsbry) (distributed along the Colorado River upflow from Blue Point Spring), albeit without support (0.70). The four sequenced specimens of P. coloradensis shared the same haplotype which differed from sequences of the other congeners included in the analysis by 4.5–11.4%. The phylogenetic analyses of theTryonia dataset congruently delineated a well-supported sister relationship between Blue Point Spring population and T. clathrata, which is also distributed in the lower Colorado River basin (Fig. 1). The Bayesian tree is shown in Fig. 3. (Note that the haplotype detected in near topotypes of T. porrecta was posi- tioned in another portion of the tree.) The depicted sister relationship between this clade and T. gilae Taylor (which is also distributed in the lower Colorado River basin) was not 112 Robert Hershler et al. / ZooKeys 492: 107–122 (2015)

P. conica (AY627958)

P. thompsoni (AY485588)

P. licina (AY367477)

P. erythropoma (AY367484)

P. milleri (GQ275096)

P. micrococcus (AY367415)

P. perforata (AY367433)

P. bacchus (DQ364005)

100 P. turbatrix (AF520936)

P. glandulosa (AY627959)

P. sola (AY627957)

P. simplex (AY627949)

P. sanchezi (AY367440) 100 P. coloradensis (PB1)

P. deserta (DQ251077)

P. kolobensis (AY627939)

P. avernalis (AF520930) 98 P. carinifera (AY627920)

P. deaconi (AY367483)

F. winkleyi (AF520917) 0.1 Figure 2. Bayesian tree based on COI data delineating the phylogenetic relationships of P. coloradensis (sequence identified by arrow). Posterior probabilities for nodes are indicated when >95%. GenBank accession numbers for haplotypes are given in parentheses. well supported. Three haplotypes (BPB-D) differing by 3–7 bps were detected in the six sequenced specimens of the Blue Point Spring Tryonia. The average divergence between these haplotypes and those of the other congeners included in the analysis was 3.9–9.0%. The Blue Point Spring population ofTryonia is morphologically diagnosable (as detailed below) in addition to being phylogenetically independent and substantially divergent genetically. We describe this distinct evolutionary lineage as a new species below. The eight sequenced specimens ofA. aff.infima shared the same haplotype which differed from sequences of the other members of theA. infima complex by 2.6 +/- 0.5%. Tajima’s relative rate test did not reject clocklike behavior for the datasets of inter- est. The posterior Bayes factor also strongly favored the molecular clock model, indicating that the application of a molecular clock is appropriate for these data. Assembly of a micro-hotspot of caenogastropod endemism in the southern Nevada desert... 113

T. infernalis (BP2C) 100 T. infernalis (BP2D) 97 T. infernalis (BP2B) T. clathrata (AF061767) T. gilae (AF12930) T. elata (AF061766) 100 T. ericae (AF061768) T. variegata (DHAF061778) T. angulata (AF061764) 100 T. contrerasi (JF776793) T. contrerasi (JF776795) 100 T. peregrina (AY803035) 100 T. pergrina (JF776804) 98 T. zaragozae (AY803038) T. allendae (AY803039) T. angosturae (AY803037) T. quitobaquitae (AF129315) T. molinae (JF776800) 100 T. taylori (JF776810) T. taylori (JF776817) 100 Minckleyella balnearis (HM149773) Minckleyella balnearis (HM149774) T. rowlandsi (AF061775) T. oasiensis (AJF776797) 100 T. chuviscarae (AJF776782) 100 T. julimesensis (AJF776789) T. minckleyi (JF776780) T. cheatumi (AF129305) T. ovata (AJF776787) T. porrecta (AF061772) 100 T. margae (AF061771) T. monitorae (AF129316) 99 T. imitator (AF061770) T. salina (AF061776) T. seemani (AJF776790) T. seemani (AJF776792) T. circumstriata (AF129306) T. metcalfi (AJF776784) T. aequicostata (AF129301) Mexipyrgus carranzae (AF129325) 0.1

Figure 3. Bayesian tree based on COI data delineating the phylogenetic relationships of the Blue Point Spring Tryonia (lineage highlighted by arrow). Posterior probabilities for nodes are indicated when >95%. GenBank accession numbers for haplotypes are given in parentheses. 114 Robert Hershler et al. / ZooKeys 492: 107–122 (2015)

Systematic description Family Cochliopidae Tryon, 1866 Genus Tryonia Stimpson, 1865

Tryonia infernalis Hershler, Liu, & Simpson, sp. n. http://zoobank.org/F7DD4C5E-E128-48AC-BAAE-866B6980C869

Undescribed [Fontelicella and] Tryonia species.—Williams et al. 1985: 32. Tryonia porrecta.—Hershler 1999: 335.

Types. USNM 883884 (a dry shell), Blue Point Spring, just below source, Clark County, Nevada, 36.3894°N, 114.4329°W, 24 July 1988, R. Hershler. Paratypes (ca. 200 dry shell and alcohol preserved specimens), USNM 1266143 (from same lot). Referred material. NEVADA. Clark County: USNM 883248 (coll. James J. Landye, 17-XII-1992), USNM 1098627 (coll. Donald W. Sada, 6-XII-2006), USNM 1146345 (coll. Andrew K. Schwaneflugel, 29-V-2008), USNM 1146420 (coll. DWS, 11-XII-2009), USNM 1248362 (coll. RH, 5–15–2014), USNM 854844 (coll. Saxon Sharpe, no date), Blue Point Spring. Diagnosis. Shell medium-sized, conic to turriform; penis having two distal papillae on the inner edge and a single basal papilla both on the inner and outer edges. Readily distinguished from geographically proximal and closely related T. clathrata by its smaller size, weaker shell sculpture, and smaller number of papillae on the inner edge of the penis. Differentiated from T. gilae (also distributed in the lower Colorado River basin) by its more convex teleoconch whorls, lateral expansion of distal bulb of penis, and in having a basal papilla on the inner edge of the penis. Differs fromT. porrecta, with which it was previously confused, by its smaller size, consistently weak shell sculpture, and much greater frequency of males. Description. Shell (Fig. 4A–B) up to 2.8 mm tall, large females having 5.00–5.75 whorls, spire height 100–133% width of shell, male shells smaller than those of females. Teleoconch whorls highly convex, evenly rounded. Aperture ovate, weakly an- gled adapically. Parietal lip complete, adnate, umbilicus narrow. Outer lip orthocline or prosocline, sometimes weakly sinuate. Sculpture of strong growth lines and a few weak spiral threads. Periostracum light brown. Shell parameters for a series of paratypes are given in Table 1. Inner and outer sides of operculum smooth (Fig. 4C–D). Radula (Fig. 4E–G): dorsal edge of central teeth concave, basal tongue V-shaped, median cusps elongate, distally pointed, lateral cusps four–six, basal cusps one–two, usually two (innermost larger; Fig. 4F). Lateral teeth having three–five cusps on inner and five–seven cusps on outer side, length of outer wing about 200% width of cutting edge, central cusp pointed (Fig. 4G). Inner marginal teeth with 24–34 cusps, outer marginal teeth with 27–38 cusps. Radula data are from USNM 1266143. Assembly of a micro-hotspot of caenogastropod endemism in the southern Nevada desert... 115

Figure 4. Shells, opercula and radula, T. infernalis sp. n. A Holotype, USNM 883884 B Male shell, USNM 1266143 C, D Opercula (outer, inner sides), USNM 1266143 E Portion of radular ribbon, USNM 1266143 F Central teeth, USNM 1266143 G Lateral and inner marginal teeth, USNM 1266143. Scale bars A–B: 1.0 mm; C, D: 200 µm; E–G: 10 µm.

Animal darkly pigmented. Penis (Fig. 5) having two distal and one basal papillae on inner edge and one basal papilla on outer edge (29 of 30 specimens); one specimen differed in having a single distal papilla along the inner edge. Distal bulb of penis ex- panded laterally on inner side, black; stylet small. Penial duct weakly undulating along most of length. Penial data are from USNM 1248362. Etymology. The specific epithet (infernalis) is a Latin adjective meaning hellish, and refers to the Valley of Fire, which is closely proximal to the type locality. 116 Robert Hershler et al. / ZooKeys 492: 107–122 (2015)

Table 1. Shell parameters for Tryonia infernalis. Measurements are in mm.

HBW/ WH SH SW HBW WBW AH AW SW/SH AH/SH SH Holotype, USNM 883884 5.75 3.09 1.78 1.87 1.56 1.13 1.00 0.58 0.604 0.36 Paratypes, USNM 1266143 (n = 9) Mean 5.33 2.61 1.41 1.60 1.25 0.95 0.80 0.54 0.61 0.37 S.D. 0.28 0.15 0.06 0.07 0.06 0.04 0.04 0.03 0.03 0.02 5.00– 2.41– 1.33– 1.46– 1.13– 0.91– 0.75– 0.49– 0.58– 0.34– Range 5.75 2.82 1.54 1.71 1.34 1.02 0.86 0.59 0.65 0.39

Figure 5. Penis (dorsal surface), T. infernalis, USNM 1248362. Scale bar: 500 µm. Db distal bulb Pa distal papilla Pd penial duct.

Distribution and habitat. Tryonia infernalis is known only from its type locality, a thermal (ca. 30 °C) rheocrene (discharging ca. 0.55 l/s; USGS 2007) whose outflow forms a narrow (ca. 0.3 m) stream (Fig. 6A). Tryonia infernalis is restricted to the upper 10 m of the spring run where it lives on silt and rocks. This species was considered to be extinct in 2002 following a series of unsuccessful searches, however it was subsequently “rediscovered” in a small, ponded reach above a weir plate associated with a USGS gaging station (Fig. 6B) in 2007 (Sada and Jacobs 2008; also see CCDCP 2002) and currently is abundant at this site (RH, personal observation). Remarks. The reproductive anatomy of several females was studied to confirm that this species belongs to Tryonia as currently defined (Hershler 2001).

Discussion

Small assemblages of locally endemic spring-dwelling invertebrates are scattered through- out arid western North America (Williams et al. 1985, Shepard 1993, Myers and Resh Assembly of a micro-hotspot of caenogastropod endemism in the southern Nevada desert... 117

Figure 6. Photographs of Blue Point Spring. A Outflow channel; spring originates below one of the mesquite trees in the upper right (photograph taken on 24/III/2009) B Ponded area where T. infernalis occurs abundantly; the USGS gage house is in the lower left (15/V/2014).

1999). Although the biogeographic history of (some of) these taxa has been studied at spatial scales ranging from local watersheds (e.g., Hershler et al. 2007b) to major hy- drographic basins (e.g., Witt et al. 2008) to the entire region (Liu and Hershler 2005), the origins of the endemic faunas of individual springs have been little investigated. The molecular phylogenetic evidence clearly points to a minimally dual origin of the Blue Point Spring fauna—T. infernalis is sister to a geographically close species from the lower Colorado River basin whereas P. coloradensis and the Blue Point Spring Assiminea are most closely related to taxa in the Death Valley region well to the west. (Note that Blue Point Spring harbors a divergent lineage of Hyalella amphipods that also is closely related to populations in the Death Valley region; Witt et al. 2006, provisional species HaPS11). The use of a molecular clock to estimate divergence times is wrought with difficul- ties and is further complicated in this case by the absence of locally derived calibrations for Assiminea and Tryonia. Nevertheless, roughly calculated values provide useful insight into the biogeographic history of the Blue Point Spring fauna (note that we performed Bayes factor and Tajima’s rate tests, both of which suggested that the assumption of a molecular clock is valid). Based on mtCOI clock calibrations of 1.83% per million years. for European Hydrobiidae (Wilke 2003) and 1.62% per m.y. for Pyrgulopsis (Hershler and Liu 2008b), the estimated divergence times of the snail populations in Blue Point Spring ranged from 1.42–2.78 Ma (Table 2). Although the age of Blue Point Spring is not known with certainty, middle to lower Pleistocene (≤2.6 Ma) spring deposits (Beard et al. 2007, map unit Q2s) provide the earliest record of local groundwater discharge. Thus, the endemic lineages may be roughly with the same age as or slightly younger than Blue Point Spring. Lake Mead and the Death Valley region are separated by the intervening, north-south trending Spring Mountains (Fig. 1) and there is no record of a prior drainage connection between these areas during the Neogene; thus it would seem likely that Assiminea and Pyrgulopsis were transported to Blue Point Spring on waterbirds. The molecular data presented here suggests that 118 Robert Hershler et al. / ZooKeys 492: 107–122 (2015)

Table 2. COI sequence divergence and estimated ages of Blue Point Spring snail lineages based on two clock calibrations.

Estimated age (m.y.) Lineage Per cent sequence divergence (sister taxon) 1.83%/m.y. calibration1 1.62%/m.y. calibration2 A. aff.infima 2.6 (other members of A. infima complex) 1.42 1.60 P. coloradensis 4.5 (P. sanchezi) 2.46 2.78 T. infernalis 3.9 (T. clathrata) 2.13 2.41 1Wilke (2003) 2Hershler and Liu (2008b) these two groups may have colonized Blue Point Spring at different times during the Pleistocene (Table 2). The sister taxon ofT. infernalis (T. clathrata) is distributed in the White River Valley, which drains into Lake Mead (via the Muddy River) a few kilometers upflow from Blue Point Spring (Fig. 1). The split between these geographically close lineages could have been a product of vicariance (e.g., severance of a thermal stream connection per Hershler et al. 1999a) or dispersal of birds (per Wesselingh et al. 1999). Whereas only a single haplotype was detected for specimens of both P. coloradensis and the Blue Point Spring Assiminea, three well differentiated haplotypes (mean divergence, 0.6%) were observed in T. infernalis, suggesting a possibly longer history of in-situ diversification. Our findings imply a relatively complex assembly of the Blue Point Spring snail fauna. The mixture of a locally derived element that may have a relatively long history of diversification within the spring Tryonia( infernalis), and lineages that appear to have colonized this water body more recently (with no sub- sequent differentiation) follows a common pattern of community assembly (Emerson and Gillespie 2008). The recognition of T. infernalis as a distinct, endemic species further highlights Blue Point Spring as a micro-hotspot of locally endemic aquatic biodiversity in Ne- vada. The Blue Point SpringAssiminea is probably a distinct species as well, but a formal taxonomic treatment is deferred pending completion of an ongoing revision of the A. infima complex (Hershler and Liu in preparation). Although this tiny aquatic ecosystem is on lands administered by the National Park Service (Lake Mead National Recreation Area), there may be a need for additional protection and conservation meas- ures. The spring is located alongside a paved highway and public access is further facilitated by a small parking area near the lower end of the spring run. There is no fencing around the spring (or its run) and thus it is vulnerable to disturbance from foot traffic and other recreational activities. [We note in this context that Blue Point Spring harbors one of the few remaining populations of the relict leopard frog (Rana onca Cope), which requires open habitat maintained by ungulate grazing and thus may be negatively impacted by fencing (Bradford et al. 2004).] The spring run appears to have been “channelized” at one time in the past, which likely resulted in a reduction of the riparian habitat utilized by Assiminea (Landye 1973). The snail fauna may be further jeop- ardized by a suite of exotic fishes that were introduced through the use of the spring as an aquarium-fish rearing establishment (until the mid-1950’s) or by aquarium release Assembly of a micro-hotspot of caenogastropod endemism in the southern Nevada desert... 119

(Deacon et al. 1964); the convict cichlid (Amatitlania nigrofasciata [Günther]), which was discovered in the spring in the 1990’s, may pose an especially serious threat owing to its omnivorous feeding habitats (Sada and Jacobs 2008). The red-rimmed melania (Melanoides tuberculata [Müller]), an invasive gastropod whose abundance appears to be negatively correlated with that of native snails in western springs based on anecdotal evidence, has also been introduced to the spring (Landye 1973).

Acknowledgements

This project was supported in part by funding from the National Park Service Mojave Desert Network Inventory and Monitoring Program (award # P13PG00412) that was facilitated by Geoffrey Moret. Sampling of Blue Point Spring snails in 2014 was made under the auspices of a scientific collecting permit from the National Park Service (Per- mit# LAKE-2014-SCI-0005). Kyle Simpson assisted with the fieldwork. The first author thanks Sue Beard for a useful discussion of the possible age of Blue Point Spring. Yolanda Villacampa measured shells and prepared scanning electron micrographs, and Freya Goetz prepared a digital version of the anatomical drawing. Ross Haley, Geof- frey Moret and Mark Sappington provided helpful comments on an early draft of this manuscript, which was also improved by input from two anonymous reviewers.

References

Abele SL (Ed.) (2011) Nevada Springs Conservation Plan. Spring Conservation Working Group. The Nature Conservancy, Reno, Nevada.http://heritage.nv.gov/sites/default/files/ library/springcons_1.pdf [accessed 8 December 2014] Beard LS, Anderson RE, Block DL et al. (2007) Preliminary geologic map of the Lake Mead 30’ × 60’ Quadrangle, Clark County, Nevada, and Mohave County, Arizona. United States Geological Survey Open-File Report 2007-1010. Plates 1–3, pamphlet. http://pubs. usgs.gov/of/2007/1010/ [accessed 17 December 2014] Bradford DF, Jaeger JR, Jennings RD (2004) Population status and distribution of a decimated amphibian, the relict leopard frog (Rana onca). Southwestern Naturalist 49: 218–228. doi: 10.1894/0038-4909(2004)049<0218:PSADOA>2.0.CO;2 Bucklin A (1992) Use of formalin-preserved samples for molecular analysis. Newsletter of Crustacean Molecular Techniques 2: 3. CCDCP (Clark County Desert Conservation Program) (2002) Biennium report for the 1999- 2001 Biennium. http://www.clarkcountynv.gov/Depts/dcp/Documents/Library/dcp%20 reports/2002/99-01BiennRptwpicturesMDH.pdf [accessed 18 December 2014] Deacon JE, Hubbs C, Zahuranec BJ (1964) Some effects of introduced fishes on the native fish fauna of southern Nevada. Copeia 1964: 384–388. doi: 10.2307/1441031 120 Robert Hershler et al. / ZooKeys 492: 107–122 (2015)

Emerson BC, Gillespie RG (2008) Phylogenetic analysis of community assembly and struc- ture over space and time. Trends in Ecology and Evolution 23: 619–630. doi: 10.1016/j. tree.2008.07.005 Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology 3: 294–299. Greenwald N, Bradley C (2008) Assessing protection for imperiled species of Nevada, U.S.A.: are species slipping through the cracks of existing protections? Biodiversity and Conserva- tion 17: 2951–2960. doi: 10.1007/s10531-008-9407-3 Hershler R (1989) Springsnails (Gastropoda: Hydrobiidae) of Owens and Amargosa River (ex- clusive of Ash Meadows) drainages, Death Valley system, California-Nevada. Proceedings of the Biological Society of Washington 102: 176–248. Hershler R (1999) A systematic review of the hydrobiid snails (Gastropoda: Rissooidea) of the Great Basin, western United States. Part II. Genera Colligyrus, Eremopyrgus, Fluminicola, Pristinicola, and Tryonia. Veliger 42: 306–337. Hershler R (2001) Systematics of the North and Central American aquatic snail genus Tryo- nia (Rissooidea: Hydrobiidae). Smithsonian Contributions to Zoology 612: 1–53. doi: 10.5479/si.00810282.612 Hershler R, Liu H-P (2008a) Phylogenetic relationships of assimineid gastropods of the Death Valley region: relicts of a late Neogene marine incursion? Journal of Biogeography 35: 1816–1825. doi: 10.1111/j.1365-2699.2008.01947.x Hershler R, Liu H-P (2008b) Ancient vicariance and recent dispersal of springsnails (Hydrobii- dae: Pyrgulopsis) in the Death Valley system, California-Nevada. In: Reheis MC, Hershler R, Miller DM (Eds) Late Cenozoic drainage history of the southwestern Great Basin and lower Colorado River region. Geological Society of America Special Paper 439: 91–101. doi: 10.1130/2008.2439(04) Hershler R, Liu H-P, Bradford C (2013) Systematics of a widely distributed North American springsnail, Pyrgulopsis micrococcus (Caenogastropoda, Hydrobiidae), with descriptions of three new congeners. ZooKeys 330: 27–52. doi: 10.3897/zookeys.330.5852 Hershler R, Liu H-P, Landye JJ (2011) New species and records of springsnails (Caenogastrop- oda: Cochliopidae: Tryonia) from the Chihuahuan Desert (Mexico and United States), an imperiled biodiversity hotspot. Zootaxa 3001: 1–32. Hershler R, Liu H-P, Mulvey M (1999a) Phylogenetic implications within the aquatic snail genus Tryonia: implications for biogeography of the North American Southwest. Molecular Phylogenetics and Evolution 13: 377–391. doi: 10.1006/mpev.1999.0659 Hershler R, Liu H-P, Sada DW (2007b) Origin and diversification of the Soldier Meadow springsnails (Hydrobiidae: Pyrgulopsis), a species flock in the northwestern Great Ba- sin, United States. Journal of Molluscan Studies 73: 167–183. doi: 10.1093/mollus/ eym014 Hershler R, Liu H-P, Thompson FG (2003) Phylogenetic relationships of North American nymphophiline gastropods based on mitochondrial DNA sequences. Zoologica Scripta 32: 357–366. doi: 10.1046/j.1463-6409.2003.00115.x Assembly of a micro-hotspot of caenogastropod endemism in the southern Nevada desert... 121

Hershler R, Liu H-P, Frest TJ, Johannes EJ (2007a) Extensive diversification of pebblesnails (Lithoglyphidae: Fluminicola) in the upper Sacramento River basin, northwestern United States. Zoological Journal of the Linnean Society 149: 371–422. Hershler R, Mulvey M, Liu H-P (1999b) Biogeography in the Death Valley region: evidence from springsnails (Hydrobiidae: Tryonia). Zoological Journal of the Linnean Society 126: 335–354. doi: 10.1111/j.1096-3642.1999.tb01375.x Hershler R, Mulvey M, Liu H-P (2005) Genetic variation in the desert springsnail (Tryonia porrecta): implications for reproductive mode and dispersal. Molecular Ecology 14: 1755–1765. doi: 10.1111/j.1365-294X.2005.02522.x Hershler R, Ratcliffe V, Liu H-P, Hay C (2014) Taxonomic revision of thePyrgulopsis gilae (Caenogastropoda, Hydrobiidae) species complex, with descriptions of two new species from the Gila River basin, New Mexico. ZooKeys 429: 69–85. doi: 10.3897/zookeys.429.7865 Landye JJ (1973) Status of the inland aquatic and semi-aquatic mollusks of the American Southwest. [Unpublished] Report to the United States Department of the Interior, Bureau of Sport Fisheries and Wildlife, Office of Rare and Endangered Species, Washington, D.C. Liu H-P, Hershler R (2005) Molecular systematics and radiation of western North American nymphophiline gastropods. Molecular Phylogenetics and Evolution 34: 284–298. doi: 10.1016/j.ympev.2004.09.013 Liu H-P, Hershler R (2007) A test of the vicariance hypothesis of western North American freshwater biogeography. Journal of Biogeography 34: 534–548. doi: 10.1111/j.1365- 2699.2006.01611.x Liu H-P, Hershler R, Clift K (2003) Mitochondrial DNA sequences reveal extensive cryptic diversity within a western American springsnail. Molecular Ecology 12: 2771–2782. doi: 10.1046/j.1365-294X.2003.01949.x Liu H-P, Hershler R, Thompson FG (2001) Phylogenetic relationships of the Cochliopinae (Rissooidea: Hydrobiidae): an enigmatic group of aquatic gastropods. Molecular Phyloge- netics and Evolution 21: 17–25. doi: 10.1006/mpev.2001.0988 Myers MJ, Resh VH (1999) Spring-formed wetlands of the arid West; islands of aquatic invertebrate biodiversity. In: Batzer DP, Rader RB, Wissinger SA (Eds) Invertebrates in freshwater wetlands of North America: ecology and management. John Wiley and Sons, New York, 811–828. Nylander JAA (2004) MrModeltest v2. Program distributed by the author. Evolutionary Biol- ogy Centre, Uppsala University. Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572–1574. doi: 10.1093/bioinformatics/btg180 Sada DW, Jacobs CA (2008) Environmental and biological characteristics of springs in Lake Mead National Recreation Area, Nevada and Arizona. Desert Research Institute, Las Ve- gas, NV. [Unpublished] Report to Lake Mead National Recreation Area, Unietd States Park Service, Boulder City, Nevada and Great Basin Cooperative Ecosystem Studies Unit. Task Agreement No. J8R07060021. http://irmafiles.nps.gov/reference/holding/146687?a ccessType=DOWNLOAD [accessed 16 December 2014] 122 Robert Hershler et al. / ZooKeys 492: 107–122 (2015)

Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4: 406–425. Shepard WD (1993) Desert springs—both rare and endangered. Aquatic Conservation: Marine and Freshwater Ecosystems 3: 351–359. doi: 10.1002/aqc.3270030409 SSI (Systat Software, Inc.) (2004) Systat® for Windows®. Richmond, CA. Swofford DL (2002) PAUP*: phylogenetic analysis using parsimony (and other methods), version 4.08b10. Sinauer Associates, Sunderland, MA. Tajima F (1993) Simple methods for testing the molecular evolutionary clock hypothesis. Genetics 135: 599–607. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution 30: 2725–2729. doi: 10.1093/molbev/mst197 Taylor DW (1987) Fresh-water molluscs from New Mexico and vicinity. New Mexico Bureau of Mines and Mineral Resources Bulletin 116: 1–50. USGS (United States Geological Survey) (2007) Water-data Report 2007. 09419547 Blue Point Spring near Valley of Fire State Park, NV. http://wdr.water.usgs.gov/wy2007/ pdfs/09419547.2007.pdf [accessed 16 December 2014] Wesselingh FP, Cadée GC, Renema W (1999) Flying high: on the airborne dispersal of aquatic organisms as illustrated by the distribution histories of the gastropod genera Tryonia and Planorbarius. Geologie en Mijnbouw 78: 165–174. doi: 10.1023/A:1003766516646 Wilke T (2003) Salenthydrobia gen. nov. (Rissooidea: Hydrobiidae): a potential relict of the Messinian salinity crisis. Zoological Journal of the Linnean Society 137: 319–336. doi: 10.1046/j.1096-3642.2003.00049.x Williams JE, Bowman DB, Brooks JE et al. (1985) Endangered aquatic ecosystems in North American deserts with a list of vanishing fishes of the region. Journal of the Arizona- Nevada Academy of Science 20: 1–62. Witt JDS, Threloff DL, Hebert PDN (2006) DNA barcoding reveals extraordinary cryptic diversity in an amphipod genus: implications for desert spring conservation. Molecular Ecology 15: 3073–3082. doi: 10.1111/j.1365-294X.2006.02999.x Witt JDS, Threloff DL, Hebert PDN (2008) Genetic zoogeography of theHyalella azteca species complex in the Great Basin: rapid rates of molecular diversification in desert springs. In: Reheis MC, Hershler R, Miller DM (Eds) Late Cenozoic drainage history of the southwestern Great Basin and lower Colorado River region. Geological Society of America Spe- cial Paper 439: 103–114. doi: 10.1130/2008.2439(05)